Nearly 50% of women worldwide develop anemia during pregnancy, and iron (Fe) deficiency remains a common problem even among otherwise well-nourished US pregnant women. Maternal Fe deficiency increases the risk of adverse birth outcomes and is increasingly recognized to impact the Fe endowment of the neonate at birth and may cause irreversible adverse effects on fetal brain development. To prevent maternal anemia, universal prenatal supplementation is recommended for all pregnant US women but recent concern has been focused on the possible risks and benefits of this practice. In order to fully address the benefits and risk of maternal Fe supplementation, a greater understanding of Fe regulation across gestation is needed. Maternal Fe absorption is regulated in response to maternal Fe stores via a complex interplay of hormones including erythropoietin, hepcidin and most recently erythroferrone (ERFE). ERFE is produced by erythroid precursors and functions to suppress hepcidin, leading to greater Fe availability for erythropoiesis. Together, these three hormones balance Fe absorption and release from stores in relation to maternal, placental and fetal Fe demands. In 2017, a new assay for ERFE was developed and extensively validated by Drs. Ganz and Nemeth. However, at this time there are no data on ERFE in human pregnancies, on how this newly identified hormone is regulated across gestation, and on how ERFE mediates the complex Fe demands and partitioning that must occur across gestation in response to maternal, placental and fetal Fe demands. The proposed research will capitalize on an existing human biospecimen archive obtained in 336 pregnant women (pregnant teens and women carrying multiple fetuses) to evaluate maternal ERFE across gestation and neonatal ERFE in umbilical cord blood in relation to maternal and neonatal Fe status, inflammation and two other key regulatory hormones (EPO and hepcidin) (Aim 1). Placental ERFE may be produced by fetal erythroid precursors to regulate neonatal Fe homeostasis and erythropoiesis. An existing placental protein and RNA archive from the neonates in Aim 1 will be used to investigate ERFE protein expression and transcript abundance between weeks 25 and 42 of gestation to identify predictors of ERFE, its association with gestational age at birth and neonatal and maternal ID and IDA. The ability of the placental-fetal unit to control neonatal Fe homeostasis will be explored using the multiplets data by evaluating variability in neonatal ERFE between siblings exposed to the same uterine environment and compared to variability observed between unrelated neonates (Aim 2). Finally, studies in wild-type and Erfe-/- pregnant females and fetuses from Fe-replete and Fe-deficient pregnancies will be undertaken to mechanistically address the contribution of ERFE to regulating erythropoiesis and Fe homeostasis in pregnancy (Aim 3). These data will provide novel information on the maternal regulatory pathways that impact Fe utilization during pregnancy and will form the basis for subsequent studies designed to improve maternal and fetal Fe status across pregnancy.